The broad objective of this project is to deduce the structural basis of protein kinase ligand selectivity and regulation. The work focuses on the casein kinase-1 (CK1) family of enzymes, which are present in all eukaryotic cells and play an essential role in regulation of the cytoskeleton. In addition to their normal physiological significance, selected members of this enzyme family are leading candidates for mediating the pathological hyperphosphorylation of cytoskeletal proteins found in neurodegenerative diseases such as Alzheimer's disease. Although members of the CK1 family are numerous and comprise one of the largest branches of the protein kinase superfamily, preliminary data suggest commonality in their mechanism of protein substrate recognition, regulation by phosphorylation, and sensitivity to small-molecule inhibitors. The goal of this proposal is to test this hypothesis by a combination of biophysical and molecular biological methods. The specific aims are: (1) to establish the basis of CK1 substrate recognition and regulation. Guided by an existing CK1 crystal structure, specific models will be tested in vitro and in vivo using oligonucleotide-directed mutagenesis; (2) to isolate and characterize a novel CK1 regulatory kinase. An enzyme that phosphorylates a site homologous to a regulatory site in cyclin-dependent protein kinases will be purified, characterized biochemically, and cloned to establish its relationship to the cyclin-dependent kinase activating kinase (CAK); and (3) to discover the mechanism of action of novel small molecule inhibitors. The binding site occupied by these molecules will be identified and sought in other protein kinases of known 3-dimensional structure. Although these experiments focus on CK1, the results will have broad implications for the protein kinase family as whole. In addition, a complete understanding of protein kinase structure and inhibitor selectivity will speed the rational development of protein kinase-selective inhibitors with potentially useful therapeutic properties.